Quantum Cascade Laser Infrared Spectroscopy for Glycan Analysis of Glycoprotein Solutions.

Glycans are oligosaccharides attached to proteins or lipids and affect their functions, such as drug efficacy, structural contribution, metabolism, immunogenicity, and molecular recognition. Conventional glycosylation analysis has relied on destructive, slow, system-sensitive methods, including enzymatic reactions, chromatography, fluorescence labeling, and mass spectrometry. Herein, we propose quantum cascade laser (QCL) infrared (IR) spectroscopy as a rapid, nondestructive method to quantify glycans and their monosaccharide composition.

Mechanism of Insoluble Aggregate Formation in a Reconstituted Solution of Spray-Dried Protein Powder.

Background: Spray-drying is considered a promising alternative drying method to lyophilization (freeze-drying) for therapeutic proteins. Particle counts in reconstituted solutions of dried solid dosage forms of biologic drug products are closely monitored to ensure product quality. We found that high levels of particles formed after reconstitution of protein powders that had been spray-dried under suboptimal conditions.

An Interlaboratory Comparison on the Characterization of a Sub-micrometer Polydisperse Particle Dispersion.

The measurement of polydisperse protein aggregates and particles in biotherapeutics remains a challenge, especially for particles with diameters of ≈ 1 µm and below (sub-micrometer). This paper describes an interlaboratory comparison with the goal of assessing the measurement variability for the characterization of a sub-micrometer polydisperse particle dispersion composed of five sub-populations of poly(methyl methacrylate) (PMMA) and silica beads. The study included 20 participating laboratories from industry, academia, and government, and a variety of state-of-the-art particle-counting instruments.

Analytical characterization of coformulated antibodies as combination therapy.

When two therapeutic agents are combined in a single formulation, i.e., coformulated, the quality and safety of the individual agents must be preserved.

Impact of Magnetic Stirring on Stainless Steel Integrity: Effect on Biopharmaceutical Processing.

Stainless steel containers are widely used in the pharmaceutical and biopharmaceutical industry for the storage of buffers, process intermediates, and purified drug substance. They are generally held to be corrosion resistant, biocompatible, and nonreactive, although it is well established that trace amounts of metal ions can leach from stainless steel equipment into biopharmaceutical products. We report here that the use of stainless steel containers in conjunction with magnetic stirring bars leads to significantly aggravated metal contamination, consisting of both metal particles and significantly elevated metal ions in solution, the degree of which is several orders of magnitude higher than described for static conditions.

Interfacial dilatational deformation accelerates particle formation in monoclonal antibody solutions.

Protein molecules are amphiphilic moieties that spontaneously adsorb at the air/solution (A/S) interface to lower the surface energy. Previous studies have shown that hydrodynamic disruptions to these A/S interfaces can result in the formation of protein aggregates that are of concern to the pharmaceutical industry. Interfacial hydrodynamic stresses encountered by protein therapeutic solutions under typical manufacturing, filling, and shipping conditions will impact protein stability, prompting a need to characterize the contribution of basic fluid kinematics to monoclonal antibody (mAb) destabilization.

Probing nucleation, reverse annealing, and chaperone function along the reaction path of HIV-1 single-strand transfer.

Reverse transcription of the HIV-1 genome involves several nucleic acid rearrangement steps that are catalyzed (chaperoned) by the nucleocapsid protein (NC), including the annealing of the transactivation response region (TAR) RNA of the genome to the complementary sequence (TAR DNA) in minus-strand strong-stop DNA. It has been extremely challenging to obtain unambiguous mechanistic details on the annealing process at the molecular level because of the kinetic involvement of a complex and heterogeneous set of nucleic acid/protein complexes of variable structure and variable composition. Here, we investigate the in vitro annealing mechanism using a multistep single-molecule spectroscopy kinetic method.

Insights on the role of nucleic acid/protein interactions in chaperoned nucleic acid rearrangements of HIV-1 reverse transcription.

HIV-1 reverse transcription requires several nucleic acid rearrangement steps that are "chaperoned" by the nucleocapsid protein (NC), including minus-strand transfer, in which the DNA transactivation response element (TAR) is annealed to the complementary TAR RNA region of the viral genome. These various rearrangement processes occur in NC bound complexes of specific RNA and DNA structures. A major barrier to the investigation of these processes in vitro has been the diversity and heterogeneity of the observed nucleic acid/protein assemblies, ranging from small complexes of only one or two nucleic acid molecules all the way up to large-scale aggregates comprised of thousands of NC and nucleic acid molecules.

Prediction of glucose in whole blood by near-infrared spectroscopy: influence of wavelength region, preprocessing, and hemoglobin concentration.

Measurement accuracy for predicting glucose in whole blood was studied based on near-infrared spectroscopy. Optimal wavelength regions, preprocessing, and the influence of hemoglobin were examined using partial least-squares regression. Spectra between 1100 and 2400 nm were measured from 98 whole blood samples.

Determination of glucose in whole blood samples by mid-infrared spectroscopy.

We have determined the glucose concentration of whole blood from mid-infrared spectra without sample preparation or use of chemical reagents. We selected 1119-1022 cm(-1) as the optimal wavelength range for our measurement by making a first-loading vector analysis based on partial least-squares regression. We examined the influence of hemoglobin on samples by using different calibration and prediction sets.

Determination of glucose concentration in a scattering medium based on selected wavelengths by use of an overtone absorption band.

A method and device for measuring glucose concentration in a scattering medium have been developed. A spectral range of 800-1800 nm is considered for wavelength selection because of its deeper penetration into biological tissue and the presence of a glucose absorption band. An algorithm based on selected wavelengths is proposed to minimize interference from other components.